The bystander cytotoxic effect, defined as the extension of the killing effects of an activated prodrug produced locally within a tumor to surrounding tumor cells, is a key feature of gene-directed enzyme prodrug therapy (GDEPT) for cancer, given that the limitation of current technologies for gene delivery allows for only a small percentage of tumor cells to be transduced with a therapeutic gene. Recent studies conducted in Dr. Waxman's laboratory suggest that a significantly enhanced bystander effect can be achieved for cytochrome P450-based GDEPT in combination with the anti-cancer prodrug cyclophosphamide by introduction of an anti-apoptotic factor, p35, into rat gliosarcoma 9L cells in a monolayer culture system. The major goals of this proposal are 1) to investigate the utility of a baculoviral caspase inhibitor, p35, in augmenting the bystander killing effect of the cancer chemotherapeutic drug cyclophosphamide in a preclinical model of cytochrome P450-based GDEPT, 2) to implement this gene therapy in a human tumor xenograft model using a replicating viral helper system, and 3) to determine whether the enhanced bystander activity seen with p35 in the P450 GDEPT system can be broadly applied to other GDEPT systems, such as herpes simplex virus thymidine kinase in combination with ganciclovir and E. coli cytosine deaminase in combination with 5-flurocytosine, both of which exert their tumor killing and/or bystander effect via distinct mechanisms. Findings obtained under this proposal may provide valuable information for paving the way of implementing this gene therapeutic strategy in the clinic.